Although it is widely accepted that B cells with self-reactivity are deleted or rendered functionally inactive, self-reactive antibodies, referred to as "natural autoantibodies," can be found in the serum of healthy animals, in an apparent paradox to the clonal tolerance theory. An anti-thymocyte/T cell autoantibody (ATA) encoded by VH36O9/VK 21C germline genes is such a natural antibody, derived from CD5+ B cells (B-1) in mice, the population predominantly responsible for natural autoantibody secretion. ATA recognizes a developmentally regulated T cell specific carbohydrate epitope on the Thy-l/CD90 glycoprotein expressed on thymocytes and helper T cells. Our previous work with VH36O9mu transgenic mice (ATAmuTg) demonstrated that the generation of ATA B cells and the secretion of serum ATA required self-antigen, since both were absent from Thy-1 knockout mice. In contrast, forced expression of ATA specificity by the bone marrow B cells in VH36O9mu/VK29C double transgenic mice (ATAmuKTg) results in predominantly negative selection as our recent study suggests. Here we propose to investigate why this positive versus negative selection occurs. By characterizing VH36O9mu/VK29C double transgenic mice (ATAmuKTg), we will identify the developmental stage(s) of B cell positive/negative selection (Aim 1). To investigate if the antigen form is critical for positive/negative selection, we will establish Thy-1 transgenic mouse lines expressing either transmembrane or secreted Thy-1 (Aim 2) In Aim 3 we will test whether positive selection is a unique feature of fetal "B-1" B cell development, selecting in favor of natural autoreactive specificities. The possibility of differences in selection threshold between "B-1" versus "B-2" B cell development will be tested (Aim 3). Accomplishing these aims will help to establish a more complete picture of antigen receptor repertoire selection in B cell development and will test our hypothesis that natural autoreactive B cell generation is a part of the innate immune system. "B-1" may actively produce certain autoreactive B cells essential for immune protection, preserving important lymphocyte clones for the rest of life to serve in immunologic surveillance.